Inflammation is a biological response to injury, infection or irritation in which a cascade of cellular and microvascular reactions serves to eradicate the infection, remove damaged tissue and generate new tissue. Immune responses mediated by various immune effectors and mediators initiate and tightly regulate the inflammatory response. However, while the normal immune system is closely regulated, aberrations in immune responses are not uncommon. Such dysregulated or excessive inflammatory reactions may result in potentially harmful processes, leading in turn to organ damage and various pathologies due to excessive exposure to inflammatory mediators or cellular effectors. These include acute pathologies such as septic shock, and chronic pathologies such as chronic gastrointestinal inflammatory diseases and autoimmune diseases.
Immune responses within the brain are specialized and differ considerably from those in the periphery. Such differences endow the central nervous system (CNS) with an immune-privilege status. Thus, the CNS is relatively secluded from the peripheral immune system, and has its own residential immune network, in which glial cells (mainly microglia and astrocytes) not only serve supportive and nutritive roles for neurons, but also defend the CNS from stress and pathogenic insults by transiently up-regulating inflammatory processes. In contrast, systemic inflammatory reactions are mediated by the peripheral immune system, and involve different leukocyte subsets, including blood borne monocytes, tissue resident macrophages, and specialized lymphocytes.
Gastrointestinal Inflammation
Crohn's disease, ulcerative colitis, inflammatory bowel disease and other related conditions form a spectrum of chronic inflammatory diseases of the gastrointestinal tract. Inflammatory bowel disease (IBD), a form of chronic gastrointestinal inflammation, includes a group of chronic inflammatory disorders including ulcerative colitis (UC) and Crohn's disease (CD). These diseases appear to result from the unrestrained activation of an inflammatory response in the intestines. This inflammatory cascade is thought to be perpetuated through the actions of pro-inflammatory cytokines and selective activation of lymphocyte subsets. In patients with IBD, ulcers and inflammation of the inner lining of the intestines lead to symptoms of abdominal pain, diarrhea, and rectal bleeding. Ulcerative colitis occurs in the large intestine, while in Crohn's the disease can involve the entire GI tract, both small and large intestines. UC is a condition that primarily affects the superficial layer of the colon mucosa and histological analyses reveal ulceration of the mucosa, blunting and loss of crypts, and an inflammatory infiltrate.
Treatment of IBD commonly utilizes a variety of orally administered systemic anti-inflammatory agents designed to reduce the inflammatory response. First line therapy commonly employs 5-aminosalicylate (Mesalamine) or its precursors, immunosuppressive agents such as cyclosporine, corticosteroids such as beclometasone and biologics such as infliximab (chimeric monoclonal antibody to tumor necrosis factor α, TNFα), anti-leukocyte adhesions molecules, and daclizumab (a recombinant humanized immunoglobulin G1 monoclonal antibody to interleukin-2 receptor α, IL-2Rα). Due to the postulated role of bacterial infection in IBD, eradication of the gut bacterial flora is also attempted by means that include use of antibiotics and antimicrobial agents. About 20-25% of the patients with UC fail to respond to medical therapy and therefore are referred to surgery for total proctocolectomy. In general, patients with CD are less responsive to medical therapy and usually do not respond to surgical treatment.
Diabetes
Diabetes mellitus is a common metabolic disorder associated with abnormally high levels of glucose in the blood. There are two major types of diabetes mellitus, termed type 1 and type 2.
Type 1 Diabetes (T1 D, or Insulin Dependent Diabetes Mellitus, IDDM) is caused by a deficiency of insulin due to an autoimmune response which leads to the destruction of the beta cells in the Islets of Langerhans of the pancreas. An initial phase of type 1 diabetes includes an inflammation of the pancreatic islets, known as insulitis. Insulitis is characterized by leukocyte and macrophage infiltration into the islets followed by the actual destruction of pancreatic beta cells in an autoimmune attack. Studies show that IDDM progresses as a predominant inflammatory beta-cell dysfunction without actual beta-cell destruction until late in the disease process.
Type 2 Diabetes (T2D, formerly non-insulin-dependent diabetes mellitus, NIDDM, or adult-onset diabetes) appears to result from both a strong genetic predisposition, and environmental factors such as diet, physical activity, and age. It is a metabolic disorder that is characterized by high blood glucose in the context of relative insulin resistance and insulin deficiency. Type 2 diabetes is caused by a combination of insulin resistance and diminished beta cell function. Insulin resistance is defined as the lack of sensitivity to insulin in adipose skeletal muscle, and hepatic tissue. As a result, the pancreas produces larger than normal amounts of insulin, a state defined as hyperinsulinemia. However, eventually the pancreas fails and insulin secretion levels decrease.
Increased levels of pro-inflammatory cytokines TNF-α, IL-1β and IL-6, are also found in the blood of diabetic patients. Inflammation may be secondary to oxidative stress induced by hyperglycemia, together with products of glycation and lipid peroxidation. A primary factor contributing to the development of insulin resistance and T2D is obesity which is characterized by a state of low-grade inflammation, in which proliferating white blood cells and activated macrophages migrate from the circulation to the tissues (Wellen et al. 2005).
Unfortunately, currently there are no cures for diabetes. Prescribed treatment generally involves control of hyperglycemia to relieve symptoms and prevent complications while minimizing hypoglycemic episodes.
Myocardial Ischemia
Heart diseases due to myocardial ischemia or ischemic heart failure are major causes of death in developed countries. When hearts are exposed to ischemia, absence of a supply of oxygen leads to the depletion of intra myocardial ATP, which if decreased by 90%, causes irreversible structural changes in the myocardium. On re-oxygenation, recovery of aerobic metabolism results in an overload of reactive oxygen species (ROS), superoxide and hydroxyl radicals which can damage cellular structures, enzymes, or channel proteins on the cellular membrane. These events can trigger the activation of inflammatory cascades and release of cytokines (Cha et al., 2008) which make cells more susceptible to myocardial contractile dysfunction or death.
Treatment for myocardial ischemia is directed at improving blood flow to the heart muscle and may include medications, a procedure to open blocked arteries or coronary artery bypass surgery. However, surgical interventions and in particular cardiac and vascular interventions possess a further risk for perioperative myocardial ischemic damage. One method for minimizing perioperative damage is known as myocardial ischemic preconditioning (IPC).
IPC is a well-established procedure for protection of the heart, especially in patients undergoing cardiac surgery. IPC consists of short ischemic period/s separated by short (re)perfusions, applied to the heart prior to prolonged ischemic insult. In humans and animal models, IPC protects the heart against injury associated with ischemia, and reduces the consequent ventricular dysfunction (Nakano et al., 2000).
Morbidity and mortality due to ischemic cardiovascular diseases are significantly higher in the elderly than in young adults. Aging causes changes in structural components of the myocardium, impairs mitochondrial function, increases monoamine oxidase (MAO)-A, and the generation of reactive oxygen species (ROS). However, hearts from aged subjects and hearts of diabetic patients are generally resistant to cardio-protection from preconditioning procedures (Przyklenk, 2011). Thus, there remains an unmet need for protecting aged subjects from myocardial damage particularly induced by cardiovascular surgery.
Endotoxin and Associated Responses
Endotoxin is invariably associated with Gram-negative bacteria. Although the term “endotoxin” is occasionally used to refer to any cell-associated bacterial toxin, in bacteriology it is properly reserved to refer to the lipopolysaccharide (LPS) complex associated with the outer membrane of Gram-negative pathogens such as Escherichia coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus influenzae, Bordetella pertussis and Vibrio cholerae. 
The biological activity of endotoxin is associated with the lipopolysaccharide (LPS). Toxicity is associated with the lipid component (Lipid A) and immunogenicity is associated with the polysaccharide components. The cell wall antigens (O antigens) of Gram-negative bacteria are components of LPS. LPS elicits a variety of inflammatory responses in animals and it activates complement by the alternative (properdin) pathway.
Specifically, LPS induces a variety of acute inflammatory responses which are qualitatively similar to those that occur during the early stages of septic shock. Moreover, LPS induces the release of a wide variety of inflammatory mediators such as pro-inflammatory cytokines (e.g. tumor necrosis factor-alpha, IL-1 beta, IL-6, IL-8), activation of the fibrinolytic system, kallikrein-kinin generation and phospholipase A2 release. Phagocytic leukocytes are primed for enhanced inflammatory responses following endotoxin administration.
Acetylcholine Esterase (AChE) Inhibitors
Acetylcholine esterase (AChE) inhibitors are a family of compounds that inhibit the breakdown of acetylcholine by the enzyme AChE, thereby increasing both the level and duration of action of the neurotransmitter acetylcholine. Reversible, AChE inhibitors Tacrine and Donepezil or pseudo-reversible AChE inhibitors such as Rivastigmine and Physostigmine, are indicated for the alleviation of neurological disorders associated with impaired acetylcholine levels, such as Alzheimer's disease (AD).
Since certain immune system cells possess various subtypes of muscarinic and nicotinic cholinergic receptors and/or synthesize AChE, it was suggested that cholinergic up-regulation may result in anti-inflammatory effects. Indeed, certain preclinical tests with acetylcholine or its agonists such as nicotine suggested immunomodulation following cholinergic up regulation (see, e.g. Borovikova et al., 2000, Wang et al., 2004). In addition, the use of nicotine patches for the treatment of human ulcerative colitis was examined in clinical trials (Sandborn et al., 1997). However, the use of nicotine as a therapeutic agent, particularly for oral use, is limited by its toxicity.
In addition, certain AChE inhibitors such as donepezil, tacrine and rivastigmine were shown to affect the immune system, particularly in the CNS. This activity was largely shown to depend on AChE inhibition and/or require their presence in the CNS. See, e.g. Nizri et al. (2006), Reale et al. (2004), Tyagi et al. (2007) and Langley et al. (2004). U.S. Patent Application Publication No. 2005/0222123 is directed to a method of treating a subject with a cytokine-mediated inflammatory disorder comprising administering to the subject a cholinesterase inhibitor other than galantamine. WO 2009/022345 to some of the inventors of the present invention is directed to the use of rivastigmine and related phenyl carbamates for the treatment of multiple sclerosis. WO 2009/022346 to some of the inventors of the present invention relates to the use of rivastigmine and such related phenyl carbamates for treating inflammatory bowel disease and other autoimmune and chronic inflammatory gastrointestinal diseases and conditions.
However, other reports indicated that the immunomodulatory effects exerted by AChE inhibitors were not sufficient to alleviate the disease outcome, particularly outside the CNS. For example, Nizri et al. (2005) discloses bi-functional compounds consisting of the non-steroidal anti-inflammatory drug ibuprofen and pyridostigmine, found effective in an EAE mouse model. Although treatment of mice by pyridostigmine alone resulted in reduced lymphocyte proliferation, such treatment did not change disease severity. Another such bi-functional compound, namely IBU-Octyl-Cytisine, containing ibuprofen and Cytisine as the nicotinic agonist, has been described by Nizri et al. (2007), which further report that each moiety separately failed to reproduce the effect of this compound. Holmes et al. (2009) found that high levels of serum TNF-α, which were associated with the degree of baseline cognitive impairment in AD patients, was independent of concomitant cholinesterase use.
One problem associated with the use of AChE inhibitors is the high degree of side effects which develop upon oral administration. These side effects include nausea, vomiting, gastrointestinal discomfort and diarrhea. Minimizing these side effects simply by limiting the administered dose may not always be applicable, because the efficacy of the drug for which AChE inhibitory activity is necessary may be impaired at lower doses.
In addition, many AChE inhibitors are contraindicated in treatment of patients afflicted with non-neurological pathologies, due to adverse effects. For example, rivastigmine is contraindicated in diabetic patients as it can increase blood sugar and promotes loss of diabetic control. Other patient populations in which rivastigmine treatment is contraindicated are those with cardiovascular/pulmonary disease and GI disorders. Indeed, despite the knowledge of potential immunomodulatory properties of these compounds, no AChE inhibitor is currently indicated for the treatment of conditions other than neurological/neurodegenerative disorders.
Ladostigil
Ladostigil, also referred to as R(+)-6-(N-methyl, N-ethyl-carbamoyloxy)-N′-propargyl-1-aminoindan and (3R)-3-(prop-2-ynylamino)-2,3,-dihydro-1H-indan-5-ylethyl methyl carbamate, is a propargyl-aminoindan with a carbamate moiety. Designed to combine the MAO-B inhibitory activity of rasagiline with AChE inhibitory activity of rivastigmine, ladostigil inhibits AChE and both MAO-A and B selectively in the brain. At much lower doses than those that inhibit either enzyme in vivo ladostigil has neuroprotective activity associated with a reduction of oxidative stress and microglial activation, neither of which is related to the ability to inhibit MAO or AChE.
Salts of ladostigil include the ½ L-tartrate salt of ladostigil. This tartrate salt of ladostigil, ladostigil tartrate-6-(N-ethyl, N-methyl carbamyloxy)-N-propargyl-1(R)-aminoindan, tartaric acid (2:1) abbreviated as [(R)-CPAI] tartrate and also referred to as ladostigil tartrate, has CAS registry number 209394-46-7 and may be used as the active ingredient of ladostigil tablets.
Ladostigil's activities include the inhibition of AChE and the inhibition of MAO. In vivo, ladostigil inhibits both MAO-A and MAO-B selectively in the brain. These activities make ladostigil particularly useful in the treatment of Alzheimer's disease comorbid with dementia.
U.S. Pat. Nos. 6,251,938, 6,303,650, and 6,538,025, incorporated herein by reference, disclose ladostigil and other compounds that inhibit AChE and MAO selectively in the brain. These compounds may be useful to treat Alzheimer's disease and other dementias such as senile dementia, dementia of the Parkinson's type, vascular dementia and Lewy body dementia, in addition to depression.
U.S. Pat. No. 7,335,685 and U.S. Application Publication Nos. 20060189819, 20070088082 and 20070093549, incorporated herein by reference, disclose crystalline forms of ladostigil tartrate and methods of producing the same. U.S. Pat. Nos. 7,375,249 and 7,476,757, and U.S. Application Publication No. 20060199974, incorporated herein by reference, disclose synthesis of enantiomeric indanylamine derivatives including ladostigil. U.S. Pat. No. 7,491,847 and U.S. Application Publication No. 20070112217, incorporated herein by reference, disclose methods for isolating propargylated aminoindans. U.S. Application Publication No. 20070203232, incorporated herein by reference, discloses methods for isolating propargylated aminoindans and discloses their use in the treatment of Alzheimer's disease. U.S. Application Publication No. 20070232691, incorporated herein by reference, discloses the use of ladostigil to treat schizophrenia.
U.S. Application Publication No. 20060189685, incorporated herein by reference, discloses formulations comprising ladostigil. U.S. Application Publication Nos. 20070135518 and 20070293583, incorporated herein by reference, discloses the use of low doses of ladostigil for neuroprotection.
WO 2005/051371 discloses a method for treatment of a subject susceptible to or suffering from a cardiovascular disorder or disease which comprises administering to the subject an amount of an active agent selected from the group consisting of propargylamine, a propargylamine derivative, and a pharmaceutically acceptable salt thereof, effective to treat the subject. Ladostigil is identified as an N-propargyl-1-aminoindan analog.
WO 2006/130726 discloses methods for the treatment of a form of multiple sclerosis comprising administering an amount of R(+)-6-(N-methyl, N-ethyl-carbamoyloxy)-N′-propargyl-1-aminoindan or a pharmaceutically acceptable salt thereof. WO '726 further discloses that ladostigil doses that were effective in ameliorating EAE (51 and 70.1 mg/kg/day) exerted 47-52% inhibition of AChE in brain and 60-65% inhibition of AChE in blood, while lower doses were not found to be effective.
WO 2012/059920, published after the priority date of the present application, relates to methods of treating individuals who have been identified as having Alzheimer's disease and other neurodegenerative diseases comprising administration of ladostigil or a pharmaceutically active salt thereof in a dosage in the range of 60-200 mg ladostigil per day.
Panarsky et al. (2010) discloses that in vitro treatment by ladostigil and three of its active metabolites decreased the release of NO from microglial cell cultures induced by LPS. The disclosure suggests that the both the carbamate moiety and propargyl moiety might not be necessary for their protective activity against LPS in microglia. The amounts of mRNA of IL-1β and TNFα in response to LPS were also reduced following ladostigil treatment.
Panarsky et al. (2012), published after the priority date of the present application, disclose that ladostigil (1 mg/kg/day) significantly decreased the gene expression of IL-1β, IL-6, TNF-α and inducible NO synthase (iNOS) in the parietal cortex. It was also shown that in vitro treatment by ladostigil and three of its active metabolites inhibited the release of NO induced by LPS from mouse microglial cells and reduced TNF-α mRNA and protein and IL-1β and iNOS mRNA.
However, none of the art discloses or fairly suggests that ladostigil may be useful as a therapeutic drug for non-neurological indications, such as for treating inflammatory diseases in peripheral organs outside the central nervous system. There remains a need for methods and formulations of ladostigil and pharmaceutically acceptable salts thereof which can effectively inhibit inflammation without the devastating side effects associated with marketed anti-inflammatory drugs. In particular, there remains an unmet medical need for therapies for pathologies associated with systemic inflammation, including e.g. gastrointestinal inflammation and inflammation associated with type 2 diabetes. Means for protecting against ischemic damage induced e.g. by surgical intervention is an unmet need.